Being the third leading cause of death and the most common cause of disability, stroke has a great effect on public health. Therefore, the search for effective treatments to prevent and/or reduce brain injury after ischemia has been a major research endeavor. Although multiple pathogenic mechanisms have been implicated in ischemic brain injury, the production of reactive oxygen species by the ischemic tissue itself during reperfusion has been recognized as a major factor initiating a series of cellular events that eventually leads to inflammation and neuronal death. Recent studies have implicated the plasma membrane NADPH oxidase in oxidative injury and our studies using post- ischemic administration of apocynin have shown that the inhibition of NADPH oxidase activity is neuroprotective in stroke. Apocynin derives from the rhizome of Picrorhiza kurroa, a well-known herb in traditional Ayurvedic medicine. It effectively inhibits NADPH oxidase through preventing the assembly of its subunits. We demonstrate that apocynin can reduce ischemia-induced lipid peroxidation and attenuate neuronal death. However, the mechanisms underlying these effects are unknown. The specific aims of this proposal are (1) to determine whether dietary preventative or post-ischemic administration of apocynin provides a better protection against ischemia-induced brain damage and behavioral deficit, and (2) to identify distinct molecular and cellular mechanisms involved in the neuroprotective effect. Studies will use a rat global cerebral ischemia model and assess neuronal cell survival and glial cell activation by histology and immunohistochemistry. Behavioral studies will evaluate whether changes in these parameters are correlated with improvement in functional outcome using a radial arm maze and an inhibitory avoidance test. Bioavailability assays will detect apocynin and diapocynin in plasma, liver and brain. The effects of apocynin on neuronal apoptosis and activation of proinflammatory responses in glial cells will be tested by quantifying superoxide production, cytochrome c release, caspase-3 activation, and changes in the expression of inflammatory markers including NADPH oxidase subunits, COX-2 and iNOS at different time points after ischemia. These studies could provide new insights on the role of NADPH oxidase and its downstream effectors pathways in ischemic injury, and will further promote testing natural products in their therapeutic pathways in animal models. PUBLIC HEALTH RELEVANCE: Stroke is the third leading cause of death in the US and the most common cause of disabilities in aging adults. In view of the fact that no conclusive evidence exists for long- lasting cognitive or motor improvement with the current drugs, the Public Health value of efficacious and safe solutions is obvious.